Detection of misfolded protein aggregates from a clinical perspective

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List of abbreviations

Alzheimer’s Disease, AD; Amyloid Precursor Protein, APP; Beta-Amyloid, Aβ; Bovine Spongiform Encephalopathy, BSE; Creutzfeldt-Jakob Disease, CJD; Central Nervous System, CNS; Immunofluorescence, IF; Immunoprecipitation, IP; Enzyme Linked Immunosorbent Assay, ELISA; Huntingtin, Htt; Huntington’s Disease, HD; Immunohistochemistry, IHC; Luminescent Conjugated Oligothiophenes, LCOs; Parkinson’s Disease, PD; Prion Protein Cellular, PrPC; Prion Protein Scra- pie-associated, PrPSc; Protein Misfolding Disease, PMD; α-Synuclein, α-Syn; Western Blot, WB.

§These authors contributed equally to this work.

*Corresponding author:

Øyvind Halskau

Department of Molecular Biology, University of Bergen, Thormøhlensgate 55, 5008, Bergen, Norway.

Tel: 004755584563

E-mail: [email protected]

Journal of Clinical and Translational Research

Journal homepage: http://www.jctres.com/en/home

REVIEW

Detection of misfolded protein aggregates from a clinical perspective

Øyvind Strømland^§, Martin Jakubec^§, Samuel Furse, Øyvind Halskau^*

Department of Molecular Biology, University of Bergen, Bergen, Norway

A R T I C L E I N F O A B S T R A C T

Article history:

Received: December 24, 2015 Revised: March 22, 2016 Accepted: March 22, 2016 Published online: March 22, 2016

Neurodegenerative Protein Misfolding Diseases (PMDs), such as Alzheimer’s (AD), Parkinson’s (PD) and prion diseases, are generally difficult to diagnose before irreversible damage to the central nervous system damage has occurred. Detection of the misfolded proteins that ultimately lead to these conditions offers a means for providing early detection and diagnosis of this class of disease. In this review, we discuss recent developments surrounding protein misfolding diseases with emphasis on the cytotoxic oligomers implicated in their aetiology. We also discuss the relationship of misfolded proteins with biological membranes. Final- ly, we discuss how far techniques for providing early diagnoses for PMDs have advanced and describe promising clinical approaches. We conclude that antibodies with specificity towards oligomeric species of AD and PD and lectins with specificity for particular glycosylation, show promise. However, it is not clear which approach may yield a reliable clinical test first.

Relevance for patients: Individuals suffering from protein misfolding diseases will likely benefit form earlier, less- or even non-invasive diagnosis techniques. The current state and possible future directions for these are subject of this review.

Keywords:

protein misfolding Alzheimer's Parkinson's

neurodegenerative disease oligomer toxicity membrane porosity lipid

antibody lectin

mass spectrometry

1. Introduction

The oligomerization and then fibrillation of misfolded pro- teins is a common feature of a large group of diseases referred to as protein misfolding diseases (PMDs). A subset of these diseases is known as neurodegenerative because they cause irreversible damage to the central nervous system (CNS). Sev- eral different proteins may precipitate the clinical symptoms of these conditions, several parts of the CNS may be damaged and the mechanism of the condition varies (Table 1). The best known examples of neurodegenerative PMDs include Parkin-

son’s

[1,2] and Alzheimer’s [2-4] diseases (PD and AD, re-

spectively). Prionic diseases are less common and less pre-

dictable than the others but their immediate clinical impact can

be more dramatic [5,6]. There are also a number of hereditary

conditions also counted among the neurodegenerative PMDs,

the most well- known of these being Huntington’s Disease

(HD)

[7]. While the exact nature and relevance of protein

misfolding is sometimes debated, for instance the relevance

and nature of Huntingtin (Htt) aggregation in HD [8,9], there

is agreement that misfolded, mis-aggregated or wrongly pro-

cessed proteins are the unifying feature of these conditions

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12 Strømland & Jakubec et al. | Journal of Clinical and Translational Research 2016; 2(1): 11-26

Table 1. Neurodegenerative protein misfolding diseases

Protein Misfolding Disease Aggregating protein(s) Aetiology Clinical Manifestation Pathogenic mechanism

AD Aβ, Tau [128] Acquired; age,

gene variants increase risk, see also familial forms

Dementia, language difficulties, executive dysfunction, depression, hallucina- tions, delusions, agitation, apathy, disin- hibition [128]

Depositions of Aβ plaques and Tau tangles observed. Selective loss of cho- linergic neurones, loss of synapses and neurones in the cerebral cortex, atrophy of frontal cortex cingulate gyrus, temporal lobe and parietal lobe [129]

Cerebral amyloid angiopathy Aβ, BRI2, Cystatin C, gelsolin, PrPSc, Transthy- rin [130]

Acquired; age, familial factors and familial subtypes types identified

Cerebral haemorrhage, ischemic lesions, progressive dementia [130]

Progressive deposition of amyloid protein in cerebral blood vessel walls lead- ing to degenerative vascular changes [130]

PD α-Syn, Tau [131,132] Acquired; head trauma, specific gene variants known to increase risk

REM sleep behaviour disorder, Exces- sive daytime sleepiness, hyposmia, depression, bradykinesia, rigidity, tremors, mild cognitive impairment, dyski- nesia, dysphagia, postural instability, freezing of gait, orthostatic hypotension [131]

Manifestation of Lewy bodies enriched in α-Syn. Loss of dopaminergic neurons in the substantia nigra, Neuroinflamma- tion with reactive gliosis and microgliosis [131]

Frontotemporal lobar degeneration

Tau, TDP-43, FUS, p62, ubiquitin [133]

Major genetic contributions [134]

Personality changes, behavioural disin- hibition, apathy, progressive aphasia [53]

Neuronal loss, gliosis, microvacular changes of frontal lobes, anterior temporal lobes, anterior cingulate cortex and insular cortex [133]

Huntington’s disease Htt [135] Congenital, monogenic

Mild psychotic and behavioural symptoms, progressive chorea, rigidity, dementia, dystonia, bradykinesia [135]

Gross striatal atrophy, neuronal loss in neocortex, cerebellum, hippocampus, substantia nigra, and brainstem nuclei [135]

Familial British dementia, and Familial Danish dementia

BRI2 [136] Congenital,

monogenic

Progressive cognitive impairment, spastic tetraparesis, cerebellar ataxa [137]

Amyloid angiopathy and neurofibrillary tangles (NFTs) in the hippocampus [136]

CADASIL, Cerebral auto- somal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

NOTCH3 [138] Congenital, monogenic [139]

Mood disturbances, apathy, subcortical ischemic events, migraine with aura, cognitive impairment [138]

Degeneration of smooth muscle cells in blood vessels [138]

Alexander disease GFAP [140] Sporadic; gene

variants increase risk

Macrocephaly, frontal leukodystrophy, palatal tremors, dysphagia, cognitive delays, seizures [140]

Demyelination, Rosenthal fibres in astrocytes [141]

Familial encephalopathy with neuroserpin inclusion bodies

Neuroserpin [142] Congenital, monogenic [143]

Dementia, epileptic, seizures, progressive myoclonus, dysarthria [142]

Poorly understood, encephalopathy with neuroserpin inclusion bodies [142]

Kuru PrP [144] Acquired; trans-

mitted

Cerebellar ataxia, choreifrom, athetoid movements, nystagmus, dysphasia [144]

Spongiform change, neuronal loss, as- trocytic microgliosis, kuru plaques [144]

Creutzfeldt-Jakob disease PrP [145] Acquired; trans- mitted

Dementia, myoclonus, visual or cerebellar disturbance, akinetic mutism, pyramidal or extrapyramidal signs [146]

Spongiform change, neuronal loss, gliosis [145]

Gerst-

mann-Straussler-Scheinker syndrome

PrP [144] Major genetic

contributions [147]

Cerebellar ataxia, gait abnormalities, dementia, dysarthria, ocular dysmetria, myoclonus, spastic paraparesis, parkinsonism, hyporeflexia or areflexia in lower extremities [144]

Amyloid plaques, severe to absent spongiform changes, neuronal loss, astrocyte microgliosis, variable neurofibrillary tangles [144]

Fatal familial insomnia PrP [144] Congenital, monogenic [148]

Insomnia, myoclonus, ataxia, dysarthria, dysphagia, pyramidal signs, autonomic hyperactivation [144]

Neuronal loss, astrogliosis, hypometab- olism in the thalamus and cingulate cortex [144]

Progressive supranuclear palsy

Tau [149] Acquired; head

trauma [150]

Progressive axial rigidity, vertical gaze palsy, dysarthria, dysphagia [149]

Neuronal loss, gliosis, neurofibrillary tangles affecting brainstem, basal gan- glia, diencephalon [149]

Chronic traumatic encephalopathy

Tau, TDP-43 [151] Acquired; head trauma [152]

Learning and memory impairment, ant- erograde amnesia, executive dysfunction, depression, apathy, irritability, sui- cidality, loss of impulse control, dementia, PD, dysarthria [151]

Atrophy of frontal and temporal cortices and medial temporal lobe, atrophy of the thalamus, hypothalamus and mammil- lary bodies. Thinning of the corpus callosum, pallor of the substantia nigra and locus coeruleus, cavum septum pellucidum [151]

Lytico-Bodig disease Tau [153] Acquired Global dementia, progressive aphasia, gaze palsy, parkinsonism, progressive supranuclear palsy [153]

Poorly understood, neurofibrillary tangles are found in the brain [153]

Continued on the next page

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Protein Misfolding Disease Aggregating protein(s) Aetiology Clinical Manifestation Pathogenic mechanism Meningioangiomatosis Tau [154] Acquired Epileptic seizures, haemorrhagic stroke,

anginoma, status epilepticus, generalized tonic–clonic seizures [155]

Focal lesion of the leptomeninges and underlying cerebral cortex [155]

Neuronal Ceroid Lipofuscinosis

ATP synthase subunit c, saposin A, saposin D [156]

Congenital, monogenic, subtypes exists [157]

Hypotonia, myoclonic jerks, generalized epileptic seizures, developmental regre- ssion, optic atrophy, macular degeneration, spastic tetraplegia, blindness, severe and constant microcephaly, and phar- maco-resistant epileptic seizures, myoclo- nia, ataxia, extrapyramidal signs [156]

Cerebellar and cortical atrophy, loss of pyramidal neurons and Purkinje cells, reactive astrogliosis [156]

Argyrophilic grain disease Tau [158] Acquired; old age [158]

Cognitive decline, dementia, mood imbalance, personality changes, behavioural abnormalities [158]

Argyrophilic grains in trans entorhinal cortex, entorhinal cortex, hippocampus, presubiculum, temporal cortex, orbito- frontal cortex, insular cortex, and amygdala[158]

[10]. Full recovery has not yet been observed in any patient

after damage to nerve tissue has begun. Clear and early diag- nosis of these conditions is therefore essential for informing sufferers about their condition, managing the condition where this is possible and giving appropriate palliative care. Theoret- ically, early diagnosis might help guide choice of treatment in cases where effective options are available.

The desire for both prompt diagnosis and improved medical treatments has thus encouraged research into misfolded protein aggregates and their relative PMDs. Diligent research has identified and characterised many of the individual proteins involved in these conditions. For example, β-amyloid (Aβ) [11]

and Tau in AD [12],

α-Synuclein (α-Syn) in PD [13,14] and

Htt in HD [7] are now largely accepted to have key roles in these diseases (For other protein involvement in given diseases, see

Table 1). Despite this, tests for diagnostic compounds

(biomarkers) are not in routine use for identifying any of the non-hereditary PMDs. For instance, when assessing patients for AD, clinicians have to rely on an imaging or visual data regarding symptoms and standardized tests that are sometimes combined with MRI [7]. Biopsies for detecting changes in the CNS are considered invasive procedures that are generally unsuitable for elderly patients, and thus they are usually only used for verification of the diagnosis post mortem [15]. Im- portantly, tissue damage precedes the formation of the charac- teristic insoluble fibrils that are detected in brains of AD suf- ferers. Such fibrils are not cytotoxic and their formation corre- lates only poorly with disease progression [16]. This suggests that other agents, such as oligomers of the same misfolded proteins as the fibrils, are responsible.

The common features of neurodegenerative PMDs, in which only one or two proteins appear to be defective, or at least the processing of which is defective, is an attractive target for translational research aiming to detect the condition in its early stages. One possible diagnostic tool is therefore a detec- tion system for the activity of proteases that are also involved in the progression of the disease, such as caspase-8 activation in the case of PD [17]

and β- and γ-secretase in AD [18,19].

One problem with this approach is that such processes do not

have a unique association with the diseases in question. An- other approach would be to focus on the individual misfolded proteins at an early stage, rather than the insoluble plaques and reduction in tissue volume associated with advanced stages.

Sensitive tools are required for the early detection of condi- tions where the underlying biochemical changes may be small or difficult to resolve. For example, only minute quantities of transmissible misfolded prions are required to precipitate Creutzfeldt-Jakob Disease (CJD) in humans [6], bovine spon- giform encephalopathy (BSE) and scrapie in sheep [20]. Nor is it necessarily straightforward to detect changes in protein folding, aggregation or processing in bodily fluids. Despite these challenges, methods for monitoring such changes in the relevant proteins of PMDs are of clinical interest. In this re- view, we explore recent advances in translational research fo- cused on detecting misfolded proteins in the context of early pre-fibril misfolding. We discuss these advances from both a research and a clinical perspective. We conclude with a for- ward-looking view on possible research directions.

2. Protein misfolding, oligomerisation and toxicity

Proteins pass through a fundamental process called folding

in order to obtain their functional structure [21-23]. Folding is

usually spontaneous under physiological conditions, and oc-

curs at rates that depend upon the protein’s size [24]. This

process takes a couple of hundred milliseconds for most pro-

teins [25]. Early steps in protein folding include the clustering

of hydrophobic amino acids and the expulsion of water, the

subsequent compaction of the polypeptide chain and consoli-

dation of secondary structure. Then, a re-ordering and fine-

tuning of the structural elements takes place to afford the final

tertiary structure. The folding process needs to make particular

intra-fold contacts both in its early and late stages. All these

steps are affected by the protein’s environment. Factors that

affect the outcome of a folding process and aggregation be-

haviour include solvent conditions, the presence or absence of

cofactors and metal cations [26], chaperones or other dissolved

factors, crowding from other proteins or macromolecular ag-

gregates, spatial organization and post-translational modifica-

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tion (reviews [27,28]). The fact that folding is influenced by many factors is also reflected in the cell biology of the PMDs.

The state of the prion protein associated with CJD is both translocated into the ER and glycosylated differently than its non-pathogenic counterparts [29,30]. Copper and zinc ions are implicated in both AD and PD, as there is evidence that they influence disease onset and progress in animal models and have been highlighted in clinical studies [31]. It is not entirely clear whether Cu

²⁺

and Zn

²⁺

are only involved in the misfold- ing of proteins or also in the aggregation of those proteins into fibrils [32].

Amyloid fibrils are widely recognized as a result of protein misfolding and have been observed both in vitro and

in vivo.

They are repetitive sheets in which monomers are joined by hydrogen bonds across β-strands. The long sheets are slightly twisted, with varying dimensions and crossover distances de- pending on the polypeptide involved. Cryo-electron micros-

copy has indicated that in Aβ the fibril is approximately 4 nm

and 11 nm at the narrowest and widest points respectively and has a twist crossover distance that has a mean of about 100 nm.

Comparative work on fibril morphology from Aβ and

α-Syn

indicates that they are similar, but with a degree of polymor- phism

[33-35]. A considerable range of proteins and protein

fragments can form fibrils, suggesting that the barrier to for- mation of these states is more likely to rest with time and physico-chemical conditions than amino acid sequence.

Importantly, the extent of fibril formation does not appear to correlate with disease progression and naturally occurring mu- tants associated with the early onset of PMDs do not produce more fibrils [16]. For these reasons, attention has been given to the aggregates preceding fibril formation. It has been proposed recently that the toxic oligomers are, just like fibrils, a general phenomenon that forms relatively independently of protein sequence [36]. The oligomerisation of other proteins such as calcitonin [37], α-Syn [38], Syrian hamster prion protein [39], GAP-43 and BASP1 [40] is consistent with this. The oligo- mers have since been shown to display significant toxici- ty-related effects relative both to monomers, fibrils and pro- to-fibrils

[41,42]. Furthermore, rates of their formation are

better able to account for disease-promoting mutations

[16,37,42,43]. It has also been suggested that their toxicity is

linked to membrane damage through a pore-like action [44]

and a range of pre-fibrillar oligomeric structures from a several proteins, including Aβ, Htt, prion proteins, and α-Syn, has since been investigated in this context [39,43,45,46].

3. Misfolded proteins and the lipid profile of the membrane

It is well established that membrane or peripheral proteins may affect membranes and vice versa (review

[47]). The li-

pid-dependent, differential processing of APP to Aβ is one particularly relevant example of this [48]. Moreover, PMD proteins and notably their oligomeric states have considerable effects on membrane integrity. Furthermore, tissue deposits of amyloid fibrils contain lipids [49]. Imaging studies on pre-

fibrillar oligomers reveal a range of structures, some of which may have a pore-like morphology (review [50]). These are referred to as amyloid pores or sometimes annular oligomers

[51]. Whether such oligomers will go on to form mature amy-

loid fibrils exclusively is not clear, as there exists reports in which pore-like oligomers do not appear to undergo fibrilla- tion

[52]. Certain drugs can arrest fibril but not oligomer for-

mation

[53]. Even though the presence of non-fibrillar, pore-

like oligomers correlates better with toxicity and disease-pro- moting mutants than fibrils, their properties, mechanism of action and what promotes or suppresses their formation re- mains poorly understood.

Two competing hypotheses that may explain the effect of oligomers on the membrane are being researched at the mo- ment. The first suggests that oligomer toxicity is a direct result of pore formation. Examples of oligomers that may effect pore formation include those generated by islet amyloid polypeptide

[54-57], poly-glutamine [58], transthyretin [59], prion protein

fragment [60], Aβ [61], β2-microglobulin [62] and serum am- yloid A [63]. Porosity was indicated by ionic flux across re- constituted membranes, which compromises cellular homeo- stasis and membrane potential [61]. The second hypothesis being tested at present is that the oligomers cause membrane thinning rather than leakage, through a distinct pore [42,64,65].

In this scenario, leakage through the membrane is independent of the pore-like aggregate morphologies and can take place through any area of the membrane that is sufficiently perturbed by these aggregates. Membrane thinning involves the increase of area per lipid and intercalation of polypeptides and water molecules between head groups in order to avoid energetically costly vacuums in the lateral lipid packing. This has the effect of lowering the dielectric barrier and allowing ion leakage through the membrane [10,66].

Experimental determination of leakage through a pore for- mation or through thinning of the membrane is not stra- ightforward. Regardless of the particular mechanism, the oli- gomers convey toxicity by perturbing the integrity of the membrane. However, the membranes may in turn affect the oligomers, too. Aggregating, oligomeric peptides have been shown to have preferential binding to particular membrane components, in particular sphingolipids and cholesterol. Sph- ingolipids and cholesterol are found in patches termed the liq- uid ordered phase that are often referred to as lipid rafts [67], though controversy about this link exists [68]. Glycosphin- golipids and gangliosides have affinity for the Aβ peptide in AD and α-Syn interacts with GM

1

and GM

3

gangliosides

[69-74]. PrP has been associated with sphingolipid signalling

platforms and bind to sphingomyelin, GalCer, GM

1

and GM

2

[70,75,76]. A strong interaction with sphingolipids may reflect

the amount of amyloidogenic protein found in possible lipid

raft areas of the extracellular leaflet of the plasma membrane

[49]. Amyloidogenic proteins such as α-Syn also interact more

strongly with anionic lipids phosphatidylglycerol and phos-

phatidylserine, found mainly in the cytoplasmic leaf

[45,77-79]. Model systems comprising the anionic lipids car-

diolipin, phosphatidic acid or phosphatidylglycerol leak more

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on contact with oligomers [45].

The role of cholesterol in membrane behaviour has been researched in some depth [80-88] but its role in oligomer for- mation and amyloidogenesis remains disputed and controver- sial

[89]. Cholesterol has been shown to bind to Aβ pro-

to-fibrils, but how these interactions influence oligomerization and later fibrillogenesis remains unclear [90-92]. There is evi- dence that cholesterol can have a stabilizing effect on mem- brane permeability as it reduces the leakage induced by α-Syn

[46]. The role of cholesterol in the proteolysis of APP to give

Aβ is better understood. Proteolysis of APP is inhibited by the group of cholesterol synthesis inhibitors known as statins

[93,94]. In PD a depletion of cholesterol leads to a decreased

level of α-Syn in membrane fractions in neuronal cell cultures and mouse brains [95]. Inhibition of cholesterol synthesis also reduces the levels of α-Syn in membranes, but the opposite applies to cholesterol supplementation in neuronal cells [96]. It has also been suggested that oxidised cholesterol accelerates aggregation of α-Syn [97].

This evidence may be at odds with the observation that cholesterol protects artificial membranes against oligomer- induced leakage [46] as it fails to provide a direct connection to the proposed toxicity mechanism. Polyunsaturated fatty acids may have an inhibitory role in oligomerization. The pre- sence of docosahexaenoic acid (DHA) suppresses the toxicity of Aβ towards SH-SY5Y cells by interfering with its aggrega- tion

[98,99], and appears to have a neuro-protective role in

murine models for AD [100]. However, DHA can also affect the progress of some cells through the cell cycle

[101]. The

notion that saturation levels of the fatty acid residues (FARs) of phospholipids in membranes play a role in modulating the rate of oligomerization agrees with measurements from model systems that indicate that saturated fatty acid residues lower the energetic barrier to aggregation [102].

Further work is required to understand the complexities of the relationship between membrane components and protein misfolding and oligomerization. It is possible that certain li- pids or other membrane components may be used as diagnostic compounds for more reliable early-stage detection of neuro- degenerative PMDs in combination with detection of the oli- gomeric proteins, should the links between lipid species and oligomerization prove robust.

4. Clinical detection of protein aggregates

There is no single rigorous assay for diagnosis of any PMD.

The mounting evidence for the involvement of toxic oligomers in neurodegeneration confers an increasing importance on de- tection methods for basic and translational research, and in clinical practice. As a result, great research effort is being fo- cused on developing clinical methods for detecting the main pathological unit of AD. At present, diagnosing AD includes a test of cognitive impairment (The Mini Mental State Exam or Folstein test), in some cases supplemented by CSF assays for phosphorylated tau and Aβ, MRI for brain volume and PET scans for Aβ plaques (or glucose metabolism) in the brain

[103].

An overview of methods for clinical detection of protein aggregates is shown in Table 2. Generally, approaches for the identification of protein aggregates can be divided into three classes of method: (i) visualization of protein aggregates in biopsies, (ii) monitoring of marker peptide in bodily fluids, and (iii) visualization of protein aggregates in vivo using im- aging techniques. Most of the methods discussed here concern

Aβ peptide detection in AD, as this field has advanced the fur-

thest. The majority of approaches rely on antibodies to confer specificity to the detection, whether it occurs in biopsies, bio-fluids or in vivo. A considerable number of different anti- bodies have been developed in the last two decades, many of which have at least some degree of specificity towards the proteins and aggregation-states involved in neurodegenerative PMDs. An overview of some of their properties is shown in

Table 3a.

The visualization of amyloid plaques in samples from biop- sies is a well-established means for qualitative detection of mature fibrils. There are several standard stains, such as Congo red and fluorescent thioflavins [104], as well as immuno- histology stains based on antibodies [105]. There have been several recent advances in the development of fluorescent probes based on luminescent conjugated oligothiophenes (LCOs) which can be used for investigating the nature of these protein deposits [106,107]. LCOs are able to illuminate more protein deposit plaques than other fluorophores [108]. Moreo- ver, the emission spectra of LCOs are dependent on the type of predominant peptide present. This makes it possible to distin- guish e.g. AD-associated aggregates from other types of ag- gregates

[109,110]. Another new and promising way of de-

tecting of amyloid plaques is the discovery of photo-induced

electron transfer probes that can be used to detect Aβ aggre-

gates without the need of a washing step [111]. These recent- ly-developed fluorescent probes represent a new opportunity in direct and sensitive identification of protein aggregates, espe- cially in complex biological environments.

The second class of methods for the detection of proteins and their aggregates detects molecules in bio-fluid samples, avoiding the need for biopsies. These clinical methods rely on the detection of marker peptides in cerebrospinal fluid (CSF).

For instance, detection of Aβ or tau protein in AD has been shown to have predictive power over which individuals will go on to develop the disease [112]. Detection of the relevant mo- lecular species in CSF is relatively straightforward and a broad array of methods exists for its detection. It is possible to detect and quantify tau peptides in the lower ng/mL range using mass spectrometry on samples acquired directly from the CNS [12], although this not in routine clinical use yet.

A more common method is the use of enzyme-linked im-

muno-sorbent assays (ELISA) [113]. For example, antibodies

2G3 and 21F12 are used for the detection of C-terminal amino

acids of Aβ peptides 1-40 and 1-42, respectively, in diagnosis

of AD [114]. The same peptides can be detected by new elec-

trochemical detection immuno-sensors. These biosensors are

based on immobilization of antibodies on gold nanostructured

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Table 2. Clinical detection of protein aggregates

Proteopathy Misfolding/oligomerizing

protein Current methods for clinical detection

AD Aβ peptide Decrease of marker peptide concentration in CSF, detected by ELISA, immu-

no-sensors [112,113,115]

MRI with plaque selective magnetic nanoparticles – hollow manganese oxide nanoparticles coated with antibody or curcumin-conjugated magnetic nanoparticles [123]

Fluorescent labelling of biotic samples with luminescent conjugated oligothiophenes [106,107,109-111]

Late phase PET imaging of cerebral fibrillary Aβ peptide with 11C-Pittsburgh compound B as a PET ligand [159,160]

Tau protein Ratio of phosphorylated Tau in position 396 and 404 in CSF could discriminate AD from other dementia; Identification by ELISA [161]

Identification of phosphorylated biomarkers pTau181, pTau199 and pTau231 in CSF by immunoassays [162-167]

Cerebral amyloid angiopathy Aβ peptide Early phase PET imaging of cerebral fibrillary β-amyloid with 11C-Pittsburgh compound B as a PET ligand [168-170]

PD α-Syn Detection of α-Syn aggregates in biotic samples by imunohistochemical or fluorescent staining [171-173]

Multi-parametric fluorescent pyrene-labelling of biotic samples [174]

Detection of α-Syn oligomers in human plasma or red cells by ELISA [175,176]

Huntington’s Disease Htt Loss of brain volume observed by MRI with combination of genetic test – identification of abnormal CAG expansion in exon1 of htt gene [177-179]

Variant Creutzfeldt-Jakob disease Prion protein PrP Whole blood immunoassay [180-182]

Other prion diseases:

Gerstmann-Sträussler-Scheinker disease, fatal familial insomnia, kuru, Creutzfeldt-Jakob Disease

Prion protein PrP Conformation dependent immunoassays in biopsy samples[183,184]

Analysis of 14-3-3 and PrPSc expression pattern in CSF [185]

Detection of PrPSc in urine by immunoassay [186,187]

screen-printed electrodes with cyclic voltammetry detection

[115], or with difference pulse voltammetry detection with

immobilization on gelsolin coated electrodes that selectively binds Aβ peptides

[116]. Another interesting approach com-

bines ELISA and surface plasmon resonance to provide greatly enhanced detection, using gold nano-particles conjugated with antibodies [117]. The role of the nano-particles in this assay is to increase the change in refractive index response that each immobilized molecule produces. This provides detection limits as low as single molecules. This technique is also designed to handle precipitates as part of the detection assay, which may be an advantage when working with oligomerization states.

The last group of methods allow direct observation of amy- loid plaques in vivo. This direct observation is appealing for clinical use, but is not yet practiced routinely. Methods like magnetic resonance imaging (MRI), positron emission tomog- raphy (PET) [118] and diffusion-tensor imaging [119,120] are being developed for direct diagnosis of amyloid plaques based on visual inspection of advanced imaging output. However, all of these methods are based only on qualitative approaches and rely on detecting visible changes in the CNS. There has been some work on quantification of amyloid loads based on PET image analysis but with very limited results [118,121]. More recent advances in MRI are based mainly on the use of parti- cles that allow localization of particular plaques. For example

it is possible to use curcumin-conjugated magnetic nano- par- ticles [122] or hollow manganese oxide nano-particles conju- gated with a particular antibody

[123]. Both of these nano-

particle methods increase the specificity and sensitivity of the techniques towards the protein aggregates. However, these approaches are not in routine use and may not satisfy the need for diagnosis before irreparable damage to tissue has occurred.

5. Concluding remarks and future perspectives

The methods available for detecting proteins associated with neurodegenerative PMDs shows some promise for clini- cal use. However, many of these methods make no distinction between monomers or oligomers. Thus, the preparation of di- agnostic tools that monitor the advancement of oligomeriza- tion at an early stage is still under development.

Antibodies are one of the most well established and still

promising directions for developing diagnostic tools. Antibod-

ies with ligand conformation sensitivity could be used to build

one or more specific standardized clinical ELISA assays for

detection of misfolded oligomers. The reliability of such im-

muno-based approaches is limited by the quality of the anti-

body involved. Notably, antibody-based detection of the mis-

folded oligomers, for instance Tau and Aβ, has advanced in

recent years (see Table 3a), indicating that standard assays

based on immunology may indeed be made sensitive to oligo-

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meric forms. Ensuring that there is a low limit of detection in a complex bio-fluid is also a concern, for ensuring early diagnosis.

Another approach could be the use of native mass spec- trometry for detecting protein aggregates. It is a technique which, in contrast to other types of mass spectrometry, can detect non-covalent interactions between proteins

[124]. This

technique is also able to detect protein complexes across a wide range of molecular masses and handles heterogeneous samples well. All these features are attractive when aiming to detect oligomers in complicated samples such as bio-fluids.

Native mass spectrometry has been used successfully to inves- tigate the assembly of virus capsid, directly from crudely puri- fied culture extract [125]. In principle, it is also possible to detect the oligomers discussed above. An overview of recent,

promising use of mass spectroscopy in neurodegenerative PMDs can be found in Table 3c.

The detection of protein glycosylation may be a means for detecting prionic diseases at an early stage (See Table 3b for references). This approach relies upon detailed knowledge of the glycosylation chemistry involved. Although mass spec- trometry may be helpful in identifying prion protein glycosyla- tion species, the most promising tool at present are lectins.

These are saccharide-binding proteins that can detect differ- ences in glycosylation with some specificity and made a dis- tinction between normal and disease-associated prionic protein successfully [126]. One limit to this approach is the ubiquity of glycosylation; it may not be clear which protein the sugars are actually attached to. For these reasons, false positive results

Table 3a. Oligomeric protein states detected by antibodies

Aggregating protein Antibody Specificity and epitope Cross-reactions Detection Methods and References Aβ-peptide 4G8 Recognizes residue 18-23 in Aβ sequence,

in its fibrils and fibrillary oligomers form

α-Syn, IAPP, Tau These cross-reactions are reported to be fibril-associated, not sequence dependent

WB, IHC, IP, ELISA [188-190]

A11 Prefibrillar oligomers, not monomers or fibrils

Weakly detects annular oligomeric con-

formations from α-Syn and IAPP WB, IHC [189,191]

6E10 Amino acid 4-9 in Aβ sequence Detects monomers, oligomers and fibrils, but does not cross-react with α-Syn or IAPP

WB, IHC, IP, ELISA, EM [189,192]

Tau T22 Human Tau oligomers; conformationally

specific epitope

Reported to have no significant cross-reaction with monomers or fibrils of Tau, or with α-Syn, IAPP, or Aβ in any form

WB, IHC, IP ELISA [193-195]

TOMA Human Tau oligomers; conformationally specific epitope

No cross-reaction with Tau monomers or

fibrils, or with Aβ or α-Syn WB ,IHC, ELISA [193]

α-Syn Syn211 Amino acid 121-125 of human α-Syn sequence

Does not cross-react with mouse or rat subtypes. Does not cross-react with β-Syn or β-Syn.

WB, IHC, IP, IF [164]

Syn-O2 Oligomers, weakly recognizes residue

127-140 of the α-Syn sequence Does not detect monomers, but detects some fibril

WB, IP, IHC [196,197]

Huntingtin (Htt) 3B5H10 PolyQ in a compact β-strand configuration Recognizes diseased-associated Htt from human and murine origin; no detectable binding to normal Htt

WB, IHC, IP, ELISA [8,198-200]

MW1 PolyQ domain of Htt exon 1 Recognizes diseased-associated Htt from human and murine origin; no detectable binding to normal Htt

WB, IHC [200,201]

Prion protein PrPC and PrPSc

6D11 PrpC, human origin; epitope within residues 93-109

Also detects PrPSc. Cross-reacts with prion proteins from cervines, ovine, murine and cricetine

WB, IHC, ELISA, IF [202,203]

G-12 Amino acid 217-232 human sequence Murine, human WB, IP, IF and ELISA [204]

PRC5 Needs Ala in position 136 PrP from murine, cervines, bovine, ovine, equine, cricetine, mustelines, sciurine, primates

WB [205]

D18 PrPC, conformationally specific epitope related to Helix 1, residues 130-160

Murine, human, recognises PrPC Only WB [206-209]

ICSM18 PrPC, conformationally specific epitope related to Helix 1, residues 130-160

Murine, human, recognises PrPC Only WB [207-209]

6H4 PrPSC conformationally specific epitope related to Helix 1, residues 130-160

Also recognises PrPC WB [207-210]

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18 Strømland & Jakubec et al. | Journal of Clinical and Translational Research 2016; 2(1): 11-26 Table 3b. Identification of prion protein glycosylation states

Prion Protein Glycosylation state Detection method Sample type References and notes Preferential detection of aglycosyl

and mono-glycosyl

Antibody PRC7; conformationally specific epitope. Residues at position 154, 166, 185 and 197 are involved.

Extract, WB The epitope is glycosylation-dependent and residues 154 and 185 are involved [205]

Sialylated and O-glycosidically linked glycans

Lectin proteins affinity for specific glycosyla- tions

Tissue, IHC Antibodies for PrP (MAB1562 and AB5058) used to ensure that lectins actually detected prion proteins [126]

Glycoproteome of prion protein variants

708 proteins or protein variants assessed Murine plasma samples Combined MS-affinity chromatography based approach [211]

Table 3c. Identification of oligomeric states by mass spectrometry

Protein Oligomeric state detected Sample type MS detection method sub-type Reference

α-Syn Differentiates between oligomers and monomers

Prepared from isolated protein Hydrogen-Deuterium Exchange, ESI-MS [212]

α-Syn Monomers and oligomers Prepared from isolated protein ESI-ion mobility mass spectrometry [213]

α-Syn Differentiates between oligomers and monomers

Conditioned cell media, similar in complexity to Cerebrospinal Fluid

Combined MS-antibody based approach [214]

APP, Prion Protein, DJ-1 Monomers and oligomers Cerebrospinal Fluid Tandem MS/MS [215]

Prion Protein Differentiates between PrPC and PrPSc

Samples prepared from brain homogenate

Quantitative LC–MS/MS [216]

may be a significant problem unless the detection method can also identify the protein involved clearly. Fortunately, many well-stablished antibodies may help solve this problem (Table

3a), although extensive glycosylation may sometimes obscure

the epitopes involved.

The problems inherent in the types of detection discussed here—low concentration of target protein, subtle differences between correctly and incorrectly folded and aggregated pro- teins, heterogeneous protein modifications and strong back- ground signals when detecting in a complex biological envi- ronment—do not have obvious solutions. However, it seems likely that protein affinity-based techniques (antibodies, lectins) can successfully be combined with instrument-based detection methods, such as mass-spectrometry, fluorescence, and surface plasmon resonance to produce sensitive detection methods able to identify both the aggregation state and modification state of the protein in question. Moreover, there is reason to believe that early detection can give the patient time to benefit from emerging medical technologies such as antibody-based inhibition of oligomer formation [127].

Disclosure

This study was funded by The Research Council of Norway, The Bergen Medical Research Foundation and the University of Bergen. The authors declare no competing interests. All contributors to the paper have read and agreed on the final version of the manuscript.

Acknowledgements

We wish to thank Dr. Kristin Viste for reading our manu- script and offering thoughtful insights and comments.

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